The activation of dendritic cells (DCs) by adjuvant molecules is essential for successful immunization. Most of the traditional and newer generations of adjuvants are derived from microbial sources and function by binding and activating Toll like receptors (TLRs) on the cell surface. We have discovered a natural adjuvant that has a uniquely potent ability to activate human and mouse DCs and can function in the absence of TLRs. This distinctive adjuvant activity is provided by Sendai virus defective genomes that are originated as byproducts of Sendai virus replication. The adjuvant activity of Sendai virus defective genomes depends on their replication by the viral polymerase. Stimulatory DI genomes do not produce viral proteins and, therefore, their activity and is not provided by encoded proteins. Defective viral genomes trigger DC maturation to the same degree as or superior to that of any TLR ligand or live virus that we have tested. Notably, unlike closely related viruses, defective viral genomes can induce the complete activation of DCs independently of complementation by type I IFN signaling and in the presence of functional viral-encoded antagonists of immunity. We hypothesize that unique cellular events are triggered by a particular viral RNA motif exposed during the replication of the defective genomes allowing for their efficient adjuvant activity. The first aim of this grant will focus on the investigation of the cellular mechanisms involved in the activation of DCs in response to defective viral particles. We propose that defective viral genomes could be used as gold standard for efficient activation of DC maturation during virus infection. The characterization of the cellular pathways involved in the response to defective viral genomes will contribute to the definition of the minimal requirements to achieve optimal DC maturation during virus infection. We will study the role of candidate cellular signaling pathways, the subcellular localization of defective viral genomes, and the association of these genomes with cellular proteins. We predict that these studies will reveal novel circuits for the activation of DCs that bypass both viral antagonism and the requirement for type I IFN signaling present during infection with standard viruses. The second aim will address the identification of the viral motifs that provide defective genomes with this distinctive activity. Based on our preliminary data, we will focus on the study of potential RNA motifs generated or exposed during the replication of the defective genome. We expect that information gathered from this aim will be significant for the elaboration of new adjuvant molecules. Finally, in the third aim we will study the immune response developed during immunization in the presence of defective viral particles and the adjuvant activity of these particles during immunization against influenza virus in mice. This study will extend our characterization of the defective virus genome's adjuvant activity in vivo and precedes the development of more elaborated methods for their harnessing and delivery. Public Health Relevance: Defective viral genomes are potent natural stimulators of the immune response that function by mechanisms distinct from those of traditional adjuvants. In this project we will investigate the cellular pathways involved in the response to infection with defective viruses and the viral motifs that trigger this response. Our goal is to obtain insight for the design of new immunostimulatory approaches and the elaboration of novel adjuvant molecules. In addition, we will test defective viral particles as adjuvants during immunization against influenza virus infection.